Process for the production of a dextran-degrading enzyme

ABSTRACT

AN IMPROVED PROCESS FOR THE PRODUCTION OF DEXTRANASE WHICH COMPRISES CULTURING A DEXTRANASE-PRODUCING MICROORGANISM SELECTED FROM THE GENERA CHAETOMIUM, HUMICOLA, SPOROTRICHUM, ANIXIELLA, MACROSPORIUM, STREPTOMYCES, GIBBERELLA, GLOESOPORIUM AND GLOMERELLA, PARTICULARLY CHAETOMIUM SPIRALE, CHAETOMIUM GRACILE, SPOROTRICHUM ASTEROIDES, OR GIBBERALLA FUJIKUORI IN A FERMENTATION MEDIUM AND RECOVERING FROM SAID MEDIUM THE DEXTRANASE WHICH ACCUMULATES THEREIN. DEXTRANASE ITSELF IS KNOWN TO BE USEFUL, FOR EXAMPLE AS ENZYMETIC PREPARATIONS FOR PREVENTION OF TOOTH DECAY OR SCALE DEPOSIT.

United States Patent 3,702,805 PROCESS FOR THE PRODUCTION OF A DEXTRAN-DEG'RADIN G ENZYME Keijiro Ishibashi, Tokio Okaniwa, Atsushi Hattori, and Masetoshi Arai, Tokyo, Japan, assiguors to Sankyo Company Limited, Tokyo, Japan No Drawing. Continuation of application Ser. No. 863,718, Oct. 3, 1969. This application Dec. 15, 1971, Ser. No. 208,389 Claims priority, application Japan, Oct. 3, 1968, 43/72,026; Nov. 5, 1968, 43/80,871; Jan. 24, 1969, 44/5,108; May 10, 1969, 44/36 ,004 Int. Cl. C0 7g 7/02 US. Cl. 195-66 R Claims ABSTRACT OF THE DISCLOSURE An improved process for the production of dextranase which comprises culturing a dextranase-producing microorganism selected from the genera Chaetomium, Humicola, Sporotrichum, Anixiella, Macrosporium, Streptomyces, Gibberella, Gloeosporium and Glomerella, particularly Cha tomium spirale, Chaetomium gracile, Sporotrichum asteroides, or Gibberella fujikuroi in a fermentation medium and recovering from said medium the dextranase which accumulates therein.

Dextranase itself is known to be useful, for example, as enzymatic preparations for prevention of tooth decay or scale deposit.

This application is a continuation of Ser. No. 863,718 filed Oct. 3, 196 9.

This invention relates to a process for the production of a dextran-degrading enzyme, dextranase, by the cultivation of certain microorganisms, the particular preference of which we found.

'Dextran partially hydrolyzed with dextranase has been used in the preparation of blood serum substitutes. Recently, dextranase has found several applications (e.g. as enzymatic preparations for prevention of tooth decay or scale deposit) in the field of dental hygiene.

Heretofore, it has been known that dextranase can be produced by the cultivation of microorganisms which belong to the genera, Penicillium, Aspergillus, Verticillium and 'Spicaria, as well as Celluvibrio. (See, for example, Acta. Chem. Scand. 3, 1405 (1949); US. Pat. 2,709,150; I. Bact. 64, 513 (1952); Acta. Chem. Scand. 2, 803 (1948); and Canadian Journal of Microbiology 3, 239 (1957 As the result of our extensive research, we have now found that certain microorganisms whose dextranaseproducing activity has been neither known nor suggested are quite effective in the large scale, biological production of dextranase.

In accordance with the present invention, there is provided a process for the production of dextranase by the cultivation of a dextranase-producing strain selected from the genera Chaetomium, Humicola, Sporotrichum, Anixiella, Macrosporium, Streptomyces, Gibberella, Gloeosporium, and Glomerella. Desired dextranase can be recovered from the culture broth in the conventional manner known per se.

Suitable microorganisms which are used as dextranaseproducing strains in the process of the invention are known ice strains and can be illustrated in the following list, in which the right column indicates the literatures showing the mycological characteristics of the micro0rganism(s) concerned. The code numbers indicated are those solely used in the applicant, Sankyo Co. Ltd., for classification.

Chaetomium:

C. spirale F-2l6-3 C. gracile F-212-8 (Fermentation Research Insgilzpte of Japan, Deposit No.

3 C. subxpirale F-2l6-4 C. bostrychodes F-203-5 Streptomyces:

S. cinnamonensz's Act 4-2 Journal of General and Applied Microbiology 6, 223-251 (1960).

Agricultural Experimental Reports, Agricultural Experiment Station of Agricultural and Commercial Department of Japan, 12, 1 110 (1898).

Hokkaido-Nolraiho (Japan) 12,

G. saubinelii F-14-l0 Gloeosporium:

G. kakz' G-12 (Fermentation Pathological and Therapeutical Research Institute of Japan, Studies on Important Parasitic Deposit N o. 308). Diseases of Persimmon" by S.

Ikata, 282 (1942), Yokendo,

Japan. Annals of the Phytopathological Society of Japan, 13, 3-4., 57

Ibid. 2, 6, 550 (1933).

G. laeticolor G-ll G. olivarum G-9 Glomerella:

G. fluctiqena G-lO (Fermentation Research Institute of Japan, Deposit No. 307).

G. lage'naria G-8 Annals of the Phytopathological Society of Japan, 16, 137 (1952).

Humicola: H. grisea F-56-4 (Fermentation Research Institute of Japan, Deposit No. 336).

Sporotrichum: S. csieroides F609 (Fermentation Research Institute of Japan, Deposit No. 337).

Anixiella: A. reticulum F-200-2 (Fermentation Research Institute of Japan, Deposit No. 335).

Macrosporium: M. bataticola F571 (Fermentation Research Institute of Japan, Deposit N o. 338).

A Manual of Soil Fungi by Gilman, 325-326 (1957).

"Pathogenic Microbiology" (part Bacteria), 956-957 (1956), Igakushoin, Japan.

Transactions of the Micrological Society of Japan, 6, 3, (1965).

Nogyo Oyobi Engei (Agriculture and Horticulture), 22, 5, p. 239 (1947).

As will be obvious for those :skilled in the art, any mutants and variants of the above-illustrated strains are also usable in the present invention.

In carrying out the present invention in practice, a suitable deXtranase-producing strain as referred to above is inoculated to a liquid or solid culture which contains various nutrient materials commonly used in an artificial or natural medium. Cultivation is then usually conducted by surface or submerged culture. Suitable nutrient materials include, for example, wheat bran, soybean meal, corn steep liquor, peptone, starch, glucose, sucrose, ammonium sulfate, urea and various other inorganic salts. Addition of l-2% dextran to the culture medium is particularly preferable because this usually increases the dextranase productivity up to ten or more times, as compared with the cultivation under non-dextran condition. Suitable cultivation temperature is about 30 C. Suitable pH may be within the range of about 5.5-7.0. Cultivation period will vary depending on other cultivation conditions but it usually is about 3 to 5 days within which a maximum potency of desired dextranase will be obtained.

Isolation and purification of desired enzyme (dextranase) from a liquid culture broth or a koji extract can be carried out in the ordinary manner which is known in the recovery of any enzyme from the fermentation broth containing the same. For example, the culture broth or aqueous koji extract obtained by the cultivation may be subjected to any one of the following steps, i.e. concentration under reduced pressure, salting out with sodium sulfate or sodium chloride, fractional precipitation with methanol, ethanol, acetone or the like solvent, adsorption-elution cycle using a suitable adsorbent material, precipitation using a protein precipitating agent, precipitation at an isoelectric point, separation of impurities with a heavy metal, electric dialysis, etc. These steps may be employed alone or in combination thereby to obtain dextranase of desired purity.

The dextran-degrading enzyme thus obtained (dextranase) can decompose dextran intensely with the result of rapid liquefaction. Its optimum effective pH is 4-6 and its optimum temperature is 55-60 C. The pH value and temperature at which dextranase is stable are 4-9 and 55 C. or lower, respectively.

The enzymatic activity of dextranase can be determined as follows: 5 ml. of a M/20 acetate buffer solution (pH 5.1) of 4.95% dextran (Dextran 2000, manufactured and sold by Pharmacia Ltd., Sweden) is added with 0.5 ml. of a suitably diluted enzyme solution and the test mixture is kept at 40 C. The time required for reducing the initial viscosity of dextran to a half (this is referred to hereafter as time of half decay) is measured. The measured value is proportional to the enzymatic activity.

Now the following examples serve to show the present invention in detail.

EXAMPLE 1 100 ml. of liquid culture medium (adjusted at pH 6.0) which contains 1% potato starch, 0.5% corn steep liquor, 0.5% peptone and 0.5% soybean meal, together with or without 1% dextran is placed in a 500 ml.-volume Erlenmeyer flask, sterilized and then inoculated with various strains specified below. Cultivation is conducted by shaking culture at 30 C. for 4 days. The dextranase enzymatic activity of the culture broth thus obtained is determined. Test results in each runs are set forth in Table 1.

TABLE 2 Time of half decay Dextran not Dextran added (exadded (ex tract with tract with Strain used 10X water) 100x water) Chaetomium spirals F-216-3 30 minutes 13 minutes Chaetomium gracile F-2l2-8 do 16 minutes.

Chaetomium subspirale F-2l6-4.. Chaetomium boxtrychodes F203-5 NoTE.Initial viscosity of the test mixture: 150 seconds; water as blank: 10 seconds.

EXAMPLE 3 TABLE 3 Time of halt decay of 0.05% enzyme sample Strain used solution, min.

Ohaetomium spirale F-2l6-3 Chaetomium gmcile F2128 Nora-Initial viscosity of the test mixture: 150 seconds; water as blank: 10 seconds.

EXAMPLE 4 100 ml. of a liquid culture medium (adjusted at pH 6.0) which contains 1% potato starch, 0.5% corn steep liquor, 0.5% peptone and 0.5% soybean meal, together with or without 1% dextran is placed in a 500 ml.-volume Erlenmeyer flask, sterilized and then inoculated with various strains specified below. Cultivation is conducted by shaking culture at 30 C. for 4 days. The dextranase enzymatic activity of the culture broth thus obtained is determined. Test results in each runs are set forth in Table 4.

TABLE 1 Time of half decay TABLE 4 g tgzgaalgot Degaeg added Time of half decay Strain used dilution) dilution) ge xegagnnot ggettaalo gtfliifiiifitiiiiiff ii k: "523? l fiififitfii Chaetomium subspimle F-zm-s 6 hours Slreptamyces cinnamonemis Act 42 4 hours 31 minutes. Chaewmium bostwchodes Rana-5' iriseoctfr eus Act i2 1- 3 dt x r Noun-Initial viscosity of the test mixture: 150 seconds; water as blank: I 0 Act 10 hour 10 seconds.

EXAMPLE 2 A solid culture medium is prepared by mixing wheat bran with its half amount of soybean meal, together with or without 2% dextran and adding an equal amount of water to the mixture, thereby forming the pasty mass. After sterilization, this solid culture medium is inoculated with various strains as specified below. Cultivation is conducted at 30 C. for 5 days according to koji culture method. The cultivation mass is extracted with water in amount of 10 or 100 parts per part of said mass. The aqueous extract is examined to determine its enzymatic activity. The text results are set forth in Table 2.

Nora-Initial viscosity of the test mixture: 50 seconds; water as blank: 10 seconds.

EXAMPLE 5 A solid culture medium is prepared by mixing wheat bran with its half amount of soybean meal, together with or without 2% dextran and adding an equal amount of water to the mixture, thereby forming the pasty mass. After sterilization, this solid culture medium is inoculated with various strains as specified below. Cultivation is conducted at 30 C. for 5' days according to koji culture method. The cultivation mass is extracted with water in amount of 10 parts per part of said mass. The aqueous extract is examined to determine its enzymatic activity. The test results are set forth in Table 5.

TABLE 5 Time of halt decay Dextran not Dextran added (exadded (extract with tract with Strain used X water) 10X water) Strepto'muces cinnamomnsis Act 4-2 hours 150 minutes.

Norm-Initial viscosity of the test mixture: 50 seconds; water as blank: 10 seconds.

EXAMPLE 6 The culture broth obtained under dextran-induced condition in Example 4 is added with acetone in amount of 3 parts by volume per part of the said broth. The re sulting precipitate is separated by filtration and then dried under reduced pressure. An enzyme sample is obtained as White powder. Yield 35% based on the weight of the culture broth. The enzymatic activity of this sample is determined with the following results.

TABLE 6 Time 01' halt decay of 0.5% enzyme sample Strain used solution, min.

Streptomyces Act 42 15 Streptomyces flavovirem Act 8-5 45 Nora-Initial viscosity of the test mixture: 50 seconds; water as blank: 10 seconds.

EXAMPLE 7 TABLE 7 Time of half decay Dextran Dextran not added added (4 times Strain used (no dilution) dilution) Humicola arisen F56-4 4 hours 100 minutes. Sporotrichum asteroids: F60-9 90 minutes minutes.

Aniziella reticulum F2002 Macrosporium bataticola F-57-1.

NOTE.Initlal viscosity of the test mixture: 150 seconds; water as blank: 10 seconds.

EXAMPLE 8 A solid culture medium is prepared by mixing wheat bran with its half amount of soybean meal, together with or without 2% dextran and adding an equal amount of water to the mixture, thereby forming the pasty mass. After sterilization, this solid culture medium is inoculated with various strains as specified below. Cultivation is conducted at 30 C. for 5 days according to koji culture method. The cultivation mass is extracted with water in amount of 10 or 100 parts per part of said mass. The aqueous extract is examined to determine its enzymatic activity. The test results are set forth in Table 8.

6 hours. Do.

TABLE 8 Time of half decay Norm-Initial viscosity of the test mixture: 150 seconds; water as blank: 10 seconds.

6 EXAMPLE 9 The cultivation mass obtained under dextran-induced condition in Example 8 is extracted with water in amount of 3 parts by volume per part of said mass. To the resulting enzyme solution, acetone is added in an amount of 3 parts by volume per part of the enzyme solution. The resulting precipitate is separated by filtration and then dried under reduced pressure. An enzyme sample is obtained as white powder. Yield 3-5 based on the total weight of the initial solid medium components. The enzymatic activity of this sample is determined with the following results.

TABLE 9 Time of hall decay 010.15% enzyme sample Strain used solution, min.

Humicola arisen F-56-4 Sporotrichum asteroides F-60-9 30 Nora.-Initial viscosity of the test mixture: 150 seconds; water as blank: 10 seconds.

EXAMPLE 10 ml. of a liquid culture medium (adjusted at pH 6.0) which contains 1% potato starch, 0.5% corn steep liquor, 0.5 peptone and 0.5% soybean meal, together with 1% dextran is placed in a 500 mL-volume Erlenmeyer flask, sterilized and then inoculated with various strains specified below. Cultivation is conducted by shaking culture at 30 C. for 4 days. The dextranase enzymatic activity of the culture broth thus obtained is determined. Test results in each runs are set forth in Table 10.

A solid culture medium is prepared by mixing wheat bran with its half amount of soybean meal, together with 2% dextran and adding an equal amount of water to the mixture, thereby forming the pasty mass. After sterilization, this solid culture medium is inoculated with various strains as specified below. Cultivation is conducted at 30 C. for 5 days according to koji culture method. The cultivation mass is extracted with water in an amount of 10 parts per part of said mass. The aqueous extract is examined to determine its enzymatic activity. The test results are set forth in Table 11.

TABLE 11 Strain used: Time of half decay Gibberella fujikuroi G-13 minutes 5 Gibberella saubinetii F14-10 hours 2 Gloeosporium kaki G-12 "do-.." 3 Gloeosporium laeticolor G-11 ..do 5 Gloeosporium olivarum G-9 ..-do 6 Glomerella fructigena G-IO do.... 6 Glomerella lagenaria G-8 do.. 12 Initial viscosity of the test mixture sec Water as blank -...-sec 10 EXAMPLE l2 The cultivation mass obtained under dextran-induced condition in Example 11 is extracted with water in amount of 3 parts by volume per part of said mass. To the resulting enzyme solution, acetone is added in an amount of 3 parts by volume per part of the enzyme solution. The

resulting precipitate is separated by filtration and then dried under reduced pressure. An enzyme sample is obtained as white powder. Yield 35% based on the total weight of the initial solid medium components. The enzymatic activity of this sample is determined with the following results. 1

TABLE 12 Enzyme sample concentration, Time oihali percent decay 0.15 30 minutes. 1.0 QOminutes. 1.0 3hours.

Strain used Gibberella J'ujikuroi G43. Gloeosporiu'm kaki G-12 Glo'merella fructiaena (3-10.

Gloeosporium laeticolor Gloeosporium olivarum Glomerella fructigena G lomerella lagenaria Humicola grisea Sporotrichum asteroides Anixiella reticulata, and

Macrosporium bataticola H g v V in a fermentation medium and recovering from said me dium the dextranase accumulated therein.

2. The process according .to claim 1 wherein said dextranase-producing microorganism is Chaetomium spirale.

3. The process according to claim 1 wherein said .dex tranase-producing microorganism is'flhaetomium gracile.

4. The process according to claim 1 wherein said 'dextranase-producing microorganism is Spbfot'richum aster,- oides.

' 5. The process according to claim lwherein said dextranase-producing microorganism is Gibberella fujikuroi.

. f e c s Cite UNITED STATES PATENTS 2,742,399 4/1956 Tsuchiya et al 195- 66 R 3,649,454 3/1972 Isono et a1. 19562 'R A. LOUIS MONACELL, Primary Examiner GJM. NATH, Assistant Examiner US. Cl. xR. 424- 1 

